Compositions and methods for treating hair follicle-related conditions

Abstract

This provides a better therapy by targeting keratinocytes and their effector molecules, which are responsible for keratin, the root sheath protein surrounding the hair shaft. [Solution] Disclosed are compositions comprising one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as the protein keratin, which assists the function and integrity of its appendages, such as hair follicles, which are assisted by keratinocytes to maintain human skin and biological barriers, function and structure. These compositions are useful for treating hair follicle-related skin diseases and conditions such as male pattern hirsutism and hidradenitis suppurativa. These compositions can also be used to loosen hair from the skin, thereby removing unwanted hair from the human body.

Description

Technical Field

[0001] The present invention includes a list of submission documents provided in an electronic format, the content of which is incorporated herein by reference.

[0002] The present invention relates to a composition for treating hair follicle-related conditions. The composition includes at least one enzyme capable of catalyzing the hydrolysis of important effector molecules such as the protein keratin or keratin-related proteins produced by keratinocytes during the process of tissue generation by keratinization. Keratinization enables keratinocytes to tether together skin layers and ensures that appendages such as hairs in the hair follicle are functional. The overproduction of said keratin during pathological keratin hyperplasia can lead to a follicular occlusion-dependent syndrome that causes various hair follicle-related skin conditions or hair follicle system skin diseases. The present invention enhances the destruction of hair root tissue, enables the hydrolysis of hair follicle proteins such as hair tethering keratin by enzymatic catalysis, enhances the removal of the entire hair shaft with significantly less force than removing untreated hair from the hair follicles of the same individual, enhances the destruction of hair root tissue within the hair follicle, and includes compositions that avoid hyperkeratosis, hair shaft abnormalities, occlusion, and the onset of skin diseases. The present invention further relates to the use of such compositions for treating, intervening in, or preventing hair follicle-related conditions and hair follicle system skin diseases including male pattern hirsutism, hirsutism, or pseudofolliculitis barbae, or acne vulgaris, keratosis pilaris, Dowling-Degos disease, Hailey-Hailey disease, mammary duct fistula, or acne conglobata, hair follicle pore disease, dissecting cellulitis of the scalp, and hidradenitis suppurativa, or for the cosmetic and aesthetic removal of unwanted hair from the skin on the human body.

Background Art

[0003] Human skin is a protective and regulatory biological barrier to the environment and is essential for homeostasis and health. Human skin has several different appendages, including nails, sweat glands, and hair follicles. The structure, function, and integrity of the skin depend on both hydrophobic lipids and keratinizing proteins. Keratinization is the process by which a subset of the protein keratin and keratin-binding proteins are paired and crosslinked by covalent and non-covalent interactions to form a structural network of fibrous and resistant tissue. The stratum corneum is the outermost layer of dead cells in the skin, mainly consisting of keratin-filled, interconnected, and dead keratinocytes, and is the main barrier against the penetration of foreign molecules. It is regulated by the level of exfoliation controlled by endogenous proteases and inhibitors. Beneath it is the more active epidermis, which consists of a large number of proliferating keratinocytes that differentiate and keratinize over time. Some stable keratinocytes constitutively express keratins such as K1, K2, K5, and K14. Others are hyperproliferating keratinocytes that, in response to external stress and disease, characteristically express keratins such as K17, K16, and the keratin 6 family (https: / / doi.org / 10.5772 / intechopen.79050).

[0004] In hair follicles, the hair shaft is formed and anchored by the inner root sheath (IRS) and outer root sheath (ORS), which allow the hair to grow from the dermal papilla. Therefore, healthy hair growth depends on the root sheath and its keratinocytes. The IRS consists of distinct sublayers: the layers of Henle, Huxley, and the cuticle (CL), which together enclose the hair shaft. The IRS gradually keratinizes through upward epidermal keratinization from the dermal papilla to the isthmus, where it desquams, facilitating the naked hair shaft to pass through the hair follicle tubule and exit the skin. Healthy hair growth is regulated by bidirectional cellular signaling between the IRS and ORS. The IRS primarily undergoes epidermal keratinization, while the ORS keratinizes through outer root sheath keratinization from the funnel just above the isthmus. Due to keratinization's central role in barrier function and integrity, many serious skin diseases of the skin and its appendages arise from dysregulated keratinization.

[0005] Those skilled in the art will recognize that follicular occlusion arises from the proliferation and dysregulated keratinization of ORS keratinocyte proteins such as keratin. Therefore, the destruction of ORS tissue at any differentiation stage by hydrolytic enzymes reaching the site of the keratosis avoids the development of the main skin conditions otherwise associated with the burden of the skin disease. The primary pathophysiological site of action of local enzymes is likely the infundibulum in most follicular skin diseases caused by ORS keratosis abnormalities. The infundibulum of the final hair follicle where most follicle-related conditions occur is located at an average depth of 500–660 μm (https: / / doi.org / 10.1007 / s00441-014-1999-1).

[0006] One model of human skin and hair follicles widely used in the development of drugs and cosmetics is, to those skilled in the art, the pig's ear, due to its similar physiological function. The mouse is an available in vivo model of hyperkeratosis, in which individual elements of the skin condition can be studied separately. Finally, since skin conditions and skin diseases are largely confined to the skin, skin biopsies from human subjects, including patients, provide the most valuable and realistic tissue models.

[0007] Hair follicles are typically distributed throughout all non-mucosal skin, but their type and appearance depend on several factors, including age, sex, and hormonal signaling. For example, mature men have predominantly coarse, colored hair growth in many areas of the body, such as the face, neck, limbs, torso, back, buttocks, and groin, whereas typically, in women, even after entering puberty, the distribution of this type of hair is not as widespread. Currently, unwanted hair removal is practiced by a large portion of the world's population, employing both physical and chemical methods. Physical devices allow for hair removal in the form of shaving, laser treatment, hair removal / epilation, or waxing. Hair removal by dissolving the hair shaft is typically performed using strong chemicals, including thioglycolates and highly alkaline solutions. Such chemicals have the disadvantage of being highly reactive, which can result in incomplete removal of the hair and hair follicles as intended, and the results may not be long-lasting, as well as causing severe skin irritation. Shaving leaves visible small hairs on the skin, causing daily skin irritation and carrying the risk of cutting. Hair removal by mechanically pulling out hair follicles is painful and inconvenient, and new hairs need to be of a certain length to be removed again. Lasers inhibit hair growth by irreversibly damaging the hair follicles, but this method requires multiple expensive treatments by a specialist, is painful, and can result in burns and scarring. This method of hair removal is only beneficial in 50% of patients treated, as its effectiveness depends directly on melanin content and is not applicable to all hair colors, skin areas, skin tones, or skin types.

[0008] Several conditions and diseases, including follicular occlusion, keratosis pilaris, Dowling-Degoss disease, Hailey-Hailey disease, mammary duct fistula, or acne clusters, pilonidal sinus disease, dissociative cellulitis of the scalp, and hidradenitis suppurativa, are associated with keratinization dysfunction. These conditions include excessive or pathological hair growth, keratosis and obstruction of the hair follicles and skin, such as follicular occlusion, keratosis pilaris, Dowling-Degos disease, Hailey-Hailey disease, mammary duct fistula, or acne clusters, pilonidal sinus disease, dissociative cellulitis of the scalp, and hidradenitis suppurativa.

[0009] Both men and women worldwide suffer from androgenetic hirsutism (Hypertrichosis). It is estimated that approximately 5-15% of women suffer from Hypertrichosis to varying degrees. Hypertrichosis is characterized by the growth of coarse, colored, masculine-patterned hair in areas of the body that are normally hairless, such as the upper lip and face, chest, back, arms, legs, groin, and buttocks. In individuals with Hypertrichosis, the symptoms are triggered by excessive signaling of the male hormone androgen, which stimulates hair follicles. In the United States alone, approximately 400,000 women have severe clinical Hypertrichosis. Self-reported Hypertrichosis clearly indicates that daily hair removal is essential for these women to lead normal and fulfilling lives. Therefore, there is a significant, yet-to-be-addressed, medical need.

[0010] One percent of the world's population suffers from hidradenitis suppurativa. Hidradenitis suppurativa is a progressive, recurrent chronic disease of the hair follicles, accompanied by multiple significant complications and a very low quality of life. In hidradenitis suppurativa and several related conditions, Hallmark's disease-triggering event is hyperkeratosis in the hair follicle infundibulum, which leads to hair follicle obstruction. In hidradenitis suppurativa, hair follicle obstruction leads to hair follicle rupture, in which cross-linked keratin aggregates leak into the dermis, which induces a severe inflammatory response, thereby resulting in deep, purulent, painful cystic lesions. These lesions are typically localized in the armpits, buttocks, groin, and skin folds, such as those found in overweight individuals and under the breasts of women. Three-quarters of patients are female. Patients typically develop the disease during puberty and have a lifelong prognosis of physical and psychological scarring and impairment. Disease severity is characterized as mild (Hurley stage 1), moderate (Hurley stage 2), and severe (Hurley stage 3), with distributions of 68%, 28%, and 4% of the patient population, respectively. In patients with moderate to severe disease, cutaneous cysts progress to sinus tunnel formation and fibrous skin scarring that impairs appearance and causes impairment.

[0011] Currently, only 800,000 patients—approximately 10% of the patient population—in the United States, Japan, Spain, Italy, France, the United Kingdom, and Germany are diagnosed with and treated for hidradenitis suppurativa, with varying levels of disease severity. Because available effective treatments are extremely limited and treatment responses are highly individualized, both diagnosed and undiagnosed patients have a significant unaddressed medical need. Treatment of exposed, sensitive skin, which can unintentionally alert others to the disease, is particularly challenging for patients, and poor treatment outcomes are likely to cause further physical and mental distress. In addition, regular hair removal is extremely difficult due to the high sensitivity of the patient's skin. First-line therapies include combinations of systemic antibiotics and hormones, and Humira®, a biologic product of an antitumor necrosis factor alpha (TNFα) monoclonal antibody, was approved by the U.S. Food and Drug Administration in 2015 on the orphan drug track for moderate to severe cases. Humira® is severely restricted in use due to its high cost, limited efficacy in managing hidradenitis suppurativa, serious and sometimes life-threatening adverse events, and numerous contraindications. In Western countries, it is estimated that 46% of patients are dissatisfied with disease management, 43% report their disease severely impacting their quality of life, and 83% have undergone skin surgery, due to a lack of access to effective treatments at present (https: / / doi.org / 10.1016 / j.jaad.2019.06.1301). Therefore, there is a need for options that enable preventive treatment and better disease management. Hyperkeratosis follicularis and its occlusion during hair growth are central bottleneck events in numerous skin conditions. Therefore, intervening in the disease at this early stage of disease development would be far more effective than existing treatments. Benzene-1,3-diol, a topical keratin-degrading chemical used to treat acne vulgaris, has shown unique but inadequate disease management benefits in several Phase II clinical trials in patients with hidradenitis suppurativa at high doses.Topical benzoyl peroxide is still in clinical trials as an option for relieving hidradenitis suppurativa.

[0012] Laser hair removal is also currently being studied in clinical trials with the aim of killing hair follicle stem cells, thereby preventing both hair growth and keratinization from the hair follicles. Initial results have shown limited preventive efficacy against hidradenitis suppurativa. In addition, this procedure requires expensive equipment, specialized skills, and certified operators to be used safely, making it inaccessible to many patients in underdeveloped and poor countries. Treating all affected areas of the body is almost always difficult due to the stinging, inflamed lesions, especially those in the private zone, where the skin is thin, difficult to touch, and sensitive.

[0013] Enzyme-based therapies, targeting keratinocytes and their effector molecules that cause root sheath proteins such as keratin surrounding the hair shaft, constitute a novel therapeutic intervention with a high potential to prevent and manage progression early, safely, and gently in a user-friendly manner, addressing unwanted hair growth, hair conditions such as male pattern hirsutism, and hair follicle-related skin diseases such as hidradenitis suppurativa and several related hair follicle-associated conditions, where there is a therapeutic or cosmetic need for better treatment. [Overview of the project]

[0014] The present invention relates to a composition comprising one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as keratin, proteins that assist in the function and integrity of human skin and biological barriers, and their appendages such as hair follicles, which are assisted by keratinocytes to maintain their function and structure. a. A pharmaceutical composition comprising at least one pharmaceutically acceptable component in addition to one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules such as keratin, a protein that assists the integrity of human skin and its appendages such as hair follicles, which are assisted by keratinocytes, or b. A cosmetic composition comprising one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as keratin, a protein that supports the integrity of human skin and its appendages, such as hair follicles, in addition to at least one cosmetically acceptable ingredient. This relates to a composition that is...

[0015] The present invention also relates to medical devices comprising the compositions of the present invention, such as medical patches with soluble microneedles embedded with enzymes, or devices with robust microneedles designed to penetrate the outermost primary skin barrier and thereby enhance the delivery of enzymes to the site of action.

[0016] The present invention further relates to the use of the compositions or medical devices of the present invention for treating hair follicle-related conditions. Use may be for medical use to treat conditions such as male pattern hirsutism, hirsutism, or pseudofolliculitis of the pubescent hairs, or hair follicle-related skin diseases induced by follicular hyperkeratosis such as pseudofolliculitis of the pubescent hairs, or other conditions of excessive or pathological hair growth caused by abnormal keratinization in the hair follicle sheath, including ORS, IRS, and hair shafts, or skin diseases induced by follicular hyperkeratosis and hair follicle obstruction, including acne vulgaris, keratosis pilaris, Dowling-Degos disease, Hailey-Hailey disease, mammary duct fistula, or Tetralogy of follicular obstruction syndrome, which includes acne vulgaris, keratosis pilaris, Dowling-Degos disease, Hailey-Hailey disease, mammary duct fistula, or acne clusters, pilonidal sinus disease, dissociative cellulitis of the scalp, and hidradenitis suppurativa, in which the same tissue is the cause of disease progression, and use may be for cosmetic and aesthetic use to remove unwanted hair from the human body.

[0017] The present invention also relates to compositions specified for use as pharmaceuticals. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 shows a screening assay for identifying the enzyme that releases hair from the skin. [Figure 2]Figure 2 shows the quantitative analysis of hair release from the skin after topical application of the enzyme composition. [Figure 3] Figure 3 shows that glutamyl endopeptidase strongly destroys the ORS tissue of human hair. [Figure 4] Figure 4 shows that glutamyl endopeptidase strongly and selectively hydrolyzes keratin derived from ORS. [Figure 5] Figure 5 shows that glutamyl endopeptidase selectively destroys healthy human ORS tissue. [Figure 6] Figure 6 shows that glutamyl endopeptidase selectively destroys ORS tissue in lesional skin of hidradenitis suppurativa. [Figure 7] Figure 7 shows that glutamyl endopeptidase promotes enhanced enzyme delivery to hair follicles. [Modes for carrying out the invention]

[0019] According to the present invention, skin diseases such as follicular skin conditions can be treated with a composition comprising one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as the protein keratin, which assists in the integrity, function, and structure of the biological barrier of human skin and its appendages, such as hair follicles.

[0020] It has been discovered that an enzyme capable of selectively and powerfully releasing hair from mammalian skin can catalyze the hydrolysis of type I and type II keratinocyte proteins such as keratin that assist in the function, integrity, function, and structure of the biological barriers of its appendages such as human skin and hair follicles, and therefore they can be used to treat human skin and / or remove or reduce hair follicle congestion to avoid skin diseases and cosmetic conditions. Thereby, the enzyme can prevent and / or reduce and / or control the progression of diseases caused by these proteins such as keratin, and inhibit the undesirable results in the progression and recurrence of skin diseases and any of their skin symptoms, without causing significant undesirable damage, and can cause an early remission of the pathological condition of the skin containing its appendages including hair follicles.

[0021] enzyme One or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as keratin, the protein that supports the integrity, function, and structure of the biological barriers of human skin and its appendages, such as hair follicles, may, in principle, be any enzyme capable of catalyzing the hydrolysis of such proteins, including those classified as keratin. Proliferative or overproliferative keratinocyte keratin, which mediates skin diseases, surrounds hair, and supports the integrity of human hair follicle tissue that promotes the natural growth of hair in its hair follicles, is typically found in human hair anchor tissue, which includes ORS or IRS, or any other clearly defined functional tissue of skin with appendages or skin with a breached barrier, and is the functional and structural protein keratin surrounding human hair that promotes the progression of the conditions or diseases described above. Those skilled in the art can determine whether a given enzyme has the ability to disrupt tissue made of proliferative or over-proliferating keratinocyte proteins, which can be classified as keratin, by simple, routine experiments such as those disclosed in Examples 1-10 and Figures 1-7. For the purposes of the present invention, an enzyme is considered to have the ability to disrupt ORS tissue in healthy hair follicles if, when its activity is measured using hairy pig skin as a substrate, compared to a negative control and an enzyme control, the reduction in force when pulling out hair after topical application is greater than 40%, preferably greater than 50%, preferably greater than 60%, preferably greater than 70%, most preferably greater than 80%, or greater than enough to avoid discernible pain during human hair removal from skin.

[0022] Preferably, one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules such as the protein keratin, which assist in the function, integrity, and structure of the biological barrier of its appendages such as human skin and hair follicles, are selected from proteases, particularly endopeptidases, especially glutamyl-specific proteases, i.e., proteases with a high specificity for cleaving peptide bonds adjacent to glutamyl residues. More preferably, one or more enzymes are selected from glutamyl endopeptidases based on their selectivity for glutamic acid amino acids in protein substrates, and keratin can avoid significant wrapping damage to the dermis. Examples of such enzymes are glutamyl endopeptidase II of Streptomyces glycerus, V8 protease of Staphylococcus aureus, particularly glutamyl endopeptidase of Bacillus, and particularly glutamyl endopeptidase ser protease bppB of Bacillus pumilus JA16 is preferred, and particularly glutamyl endopeptidase blaSE of Bacillus licheniformis is preferred.

[0023] In a preferred embodiment, one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules such as the protein keratin, which assist in the integrity, function, and structure of the biological barrier of its appendages such as human skin and hair follicles, are selected from proteases, preferably glutamyl endoproteases having at least 60% sequence identity, such as at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, to the polypeptide having the sequence of SEQ ID NO: 1 or SEQ ID NO: 13. In a preferred embodiment, one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules such as the protein keratin, which assist in the integrity, function, and structure of the biological barrier of its appendages such as human skin and hair follicles, comprise the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 13.

[0024] One or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as the protein keratin, which assists in the integrity, function, and structure of the biological barriers of human skin and its appendages, such as hair follicles, may be mutants of proteases containing the sequences of SEQ ID NO: 1 and SEQ ID NO: 13, such as mutants containing one or more substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably conservative substitutions, compared to SEQ ID NO: 1 or SEQ ID NO: 13.

[0025] In another preferred embodiment, one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as the protein keratin, which assists the integrity, function, and structure of the biological barriers of human skin and its appendages, such as hair follicles, are selected from proteases having at least 60% sequence identity, for example, at least 70% sequence identity, for example, at least 80% sequence identity, for example, at least 90% sequence identity, for example, at least 95% sequence identity, for example, at least 96% sequence identity, for example, at least 97% sequence identity, for example, at least 98% sequence identity, for example, at least 99% sequence identity, preferably glutamyl endoproteases. In a preferred embodiment, one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as the protein keratin, which assists the integrity, function, and structure of the biological barriers of human skin and its appendages, such as hair follicles, contain the sequence of SEQ ID NO: 12 and / or SEQ ID NO: 14.

[0026] One or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as the protein keratin, which assists in the integrity, function, and structure of the biological barriers of human skin and its appendages, such as hair follicles, may be mutants of proteases containing the sequences of SEQ ID NO: 12 and SEQ ID NO: 14, such as mutants containing one or more substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably conservative substitutions, compared to SEQ ID NO: 12 or SEQ ID NO: 14.

[0027] Those skilled in the art can determine whether a given enzyme has the ability to destroy ORS tissue containing keratinocytes and their effector proteins, which can be classified as keratin, by simple, routine experiments such as the experiments and results disclosed in Example 3. Preferably, the enzyme can achieve a reduction of more than 40% in the force of hair pulling when the enzyme is applied compared to the negative control, if the positive control is significantly different from the negative control. The enzyme is considered capable of destroying healthy tissue when supplied at concentrations of less than 1000 ppm, preferably less than 100 ppm, and more preferably less than 10 ppm. Particularly good concentration ranges are between 0.01 ppm and 1 ppm, and even more preferably between 0.01 ppm and 0.05 ppm.

[0028] In skin sample analysis (Examples 6-9), instead of using a negative control buffer (without added enzyme), enzyme incubation was performed using 30-1000 ppm, preferably 1-10 ppm, preferably less than 1 ppm, or even less than 0.1 ppm, more preferably 0.01-0.1 ppm, followed by histological examination under a microscope. Preferably, instead of using a negative control buffer, enzyme incubation was performed using 30-1000 ppm, preferably 1-10 ppm, preferably less than 1 ppm, or even less than 0.1 ppm, preferably 0.01-0.1 ppm. The ability of an enzyme composition to destroy ORS tissue can be observed if, after enzyme incubation, it visibly destroys ORS with plucked beard hairs, thereby causing significant swelling under a microscope, or if, instead of a negative control buffer, it destroys the epithelium using 30-1000 ppm of enzyme, preferably 1-10 ppm of enzyme, preferably less than 1 ppm of enzyme, or even less than 0.1 ppm of enzyme, preferably 0.01-0.1 ppm of enzyme, thereby visibly degrading the ORS tissue without significantly damaging the ORS cells under microscopic observation after enzyme incubation, rather than using a negative control buffer.

[0029] Those skilled in the art can determine whether a given enzyme has the ability to destroy healthy ORS tissue containing keratinocytes and their effector proteins, which can be classified as keratin, by simple, routine experiments such as those disclosed in Examples 6-8 and their results.

[0030] Those skilled in the art can determine whether a given enzyme has the ability to destroy lesional ORS tissue containing keratinocytes and their effector proteins, which can be classified as keratin, by simple, routine experiments such as those disclosed in Example 9 and their results.

[0031] Pharmaceutical composition The pharmaceutical compositions of the present invention are used for topical application to the skin site to be treated. Those skilled in the art can determine whether a given enzyme is delivered to the site of action in the hair follicle after topical application by simple, routine experiments such as the experiment and its results disclosed in Example 10. In Example 10, the enzyme enhances the delivery of gold nanoparticles to hair follicles at least 300 μm away from the skin surface. In the present invention, the enzyme is considered to have been delivered topically if, as a result of the enzyme activity, the gold nanoparticles penetrate considerably deeper into the hair follicle compared to a negative control (the same composition without the enzyme).

[0032] Therefore, the composition of the present invention is preferably in the following form. - Forms suitable for topical application, such as creams, lotions, gels, ointments, foams, films, sprays, particles, filaments, or suspensions of spheres having a size of 10-1000 nanometers, or combinations thereof that penetrate more easily and deeply into hair follicles and skin, - In some cases, in the form of an occlusive treatment that promotes skin swelling, or a patch that physically and / or chemically promotes the penetration of skin components and enzymes into an enhanced active site, such as a reservoir holding microneedles or similar pointed structures or compositions, or - Other known formulations or forms of administration that are approved or will be approved in the future for targeted application to skin or lesions, such as in the form of skin implants, for example, a physical reservoir, pump, or particle or composition in which a device such as a needle is placed.

[0033] The composition may also be formulated as an intrafollicular or intradermal injection, preferably using a hollow needle, to treat existing lesions by injecting the solution into the site of action where the causative protein of the advanced lesion, which has potentially low permeability, should be hydrolyzed.

[0034] Preferably, one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as the protein keratin, which assists the function and integrity, function, and structure of the biological barriers of human skin and its appendages such as hair follicles, are applied in an amount of 10 ng to 10,000 μg of enzyme protein per square centimeter of skin, preferably 100 ng to 1,000 μg of enzyme protein per square centimeter of skin, and more preferably 100 ng to 100 μg of enzyme protein per square centimeter of skin.

[0035] Preferably, one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as the protein keratin, which assists the function and integrity, function, and structure of the biological barriers of skin lesions such as human skin, occluded hair follicles, and cysts, are injected into the affected skin in an amount of 10 ng to 10,000 μg of enzyme protein per skin site to be treated, preferably 100 ng to 1,000 μg of enzyme protein per skin lesion, and more preferably 100 ng to 100 μg of enzyme protein per skin site to be treated.

[0036] The compositions of the present invention preferably contain one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as keratin, proteins that support the function and integrity, function and structure of the biological barriers of human skin and its appendages, such as hair follicles, in an amount ranging from 1 ng to 100 mg of enzyme protein per gram of composition, or, expressed in ppm in the present invention as 1 ppm = 1 mg of enzyme protein per L volume of the composition of the present invention, preferably in an amount of 0.01 ppm to 1000 ppm of composition, preferably in an amount of 1 ppm to 100 ppm of composition, more preferably in an amount of 30 ppm to 100 ppm of composition, in which the compositions contain one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as keratin, proteins that support the function and integrity, function and structure of the biological barriers of human skin and its appendages, such as hair follicles.

[0037] In one embodiment, the composition is a pharmaceutical composition whose components and concentrations are pharmaceutically acceptable or will be pharmaceutically acceptable in accordance with the current drug regulations in effect, or which are otherwise specifically authorized by the regulatory authority for a particular composition to be marketed and applied in the relevant market, state, or disease.

[0038] In this specification and the claims, “pharmaceutically acceptable ingredients,” “ingredients that are pharmaceutically acceptable,” or grammatically equivalent terms mean ingredients that possess the necessary homogeneity and purity for use as components in the manufacture of a pharmaceutical composition. Those skilled in the art know how to select suitable ingredients acceptable for such use, for example, by using an accepted pharmacopoeia such as the United States Pharmacopoeia, the European Pharmacopoeia, or equivalent legal and regulatory bodies worldwide.

[0039] In one embodiment, additional excipients are added to the composition in addition to at least one pharmaceutically acceptable component.

[0040] In one embodiment, the composition further comprises other pharmaceutically active molecules.

[0041] In one embodiment, the composition further comprises a pharmaceutically active molecule that relieves pain.

[0042] In one embodiment, the composition further comprises a pharmaceutically active molecule that reduces inflammation.

[0043] In one embodiment, the composition further comprises a pharmaceutically active molecule that reduces hair growth.

[0044] In one embodiment, the composition is a semi-solid composition such as an emulsion.

[0045] In one embodiment, the composition is a suspension containing a liquid or semi-solid composition in which solid or semi-solid particles, filaments, or mixtures thereof are dispersed. Preferably, the particles have an average size of 10 to 10,000 nm, preferably 50 to 2,000 nm, and particularly 100 to 800 nm.

[0046] In one embodiment, the composition is delivered locally, thus further enhancing the pharmaceutically active molecule's advantages.

[0047] Additional composition excipients The compositions of the present invention include one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules such as keratin, a protein that assists the function and integrity, function, and structure of the biological barriers of human skin and its appendages such as hair follicles, and one or more further components selected from water, pH adjusters, preservatives, protein denaturants such as chaotropic agents, organic salts, reducing agents, diluents, ionic strength adjusters, surfactants, alcohols, organic salts, chelating agents, pH-adjusting compounds, enzyme cofactors, waxes, compositions, and enzyme stabilizers, lipids including essential oils, oils, and waxes, in combination with keratin-degrading agents such as anti-inflammatory and analgesic compounds, skin peeling agents, and health microbiome-promoting substances. Preferably, one or more further components are selected from enzyme activators such as calcium salts and pH buffer salts, stabilizers such as polyols and sugars, humectants such as surfactants, chaotropic agents, reducing agents, and permeability-enhancing components such as water.

[0048] In one embodiment, the composition has additional excipients that reduce water activity by being present in the composition at a concentration of 1-99%, preferably 1-80%, preferably 1-60%, more preferably 1-40%. This is because it reduces microbial growth that could impair the composition and increases the stability of enzymes such as proteases, as their activity is water-dependent. Preferably, these excipients are selected from polyols such as glycerol and polyethylene glycol.

[0049] In one embodiment, the composition contains a surfactant. The hydrophilic-lipophilic balance (HLB) of a surfactant is a measure of the degree to which it is more hydrophilic or more lipophilic, and is known to those skilled in the art.

[0050] In one embodiment, the surfactant is cationic.

[0051] In another embodiment, the surfactant is anionic, such as sodium lauryl sulfate, to ensure wetting of the water-containing composition and enhance skin swelling, thereby promoting molecular permeability to external compounds.

[0052] In another embodiment, to ensure wetting of the water-containing composition and enhance delivery through hydrophobic barriers such as hair follicle sebum, the surfactant is a low HLB nonionic surfactant such as sorbitan laurate with a span of 20. Preferably, the nonionic surfactant has an HLB value between 0 and 20, more preferably between 5 and 12. Preferably, the nonionic surfactant has an HLB between 5 and 18, such as 0.001-10% Triton X100. Preferably, the surfactant has an HLB between 5 and 12, such as 0.01-1% Triton X100. Another suitable nonionic surfactant is a low HLB nonionic surfactant such as sorbitan laurate with a span of 20, such as 0.1-5%. Preferably, the surfactant is a low HLB nonionic surfactant such as sorbitan laurate with a span of 20, such as 0.5-2%.

[0053] In one embodiment, different surfactants are mixed to reduce the concentration of free single surfactants that may disrupt the stability of proteins such as enzymes. A good mixture is a mixture of a nonionic surfactant with a high HLB of 8-20, such as 0.01-1% Triton X100, and a nonionic surfactant with a low HLB of 0-12, such as sorbitan laurate with a Span of 20, such as 0.1-5% Span 20. Another good mixture is a combination of a nonionic surfactant and an anionic surfactant with low water activity.

[0054] In one embodiment, the composition further comprises a buffer salt, such as 10–1000 mM Tris / HCl, to ensure a stable pH within the range where one or more enzymes in the composition are most active. In a preferred embodiment, the composition comprises a buffer salt, such as 25–200 mM Tris / HCl, to ensure a stable pH within the range where one or more enzymes in the composition are most active, allowing the composition to limit potential skin irritation under near physiological conditions.

[0055] In one embodiment, the composition further comprises salts that allow the composition to limit potential skin irritation, such as 0.01–2% NaCl, which is close to physiological conditions. In a preferred embodiment, the composition comprises salts that adjust the ionic strength to a more physiologically optimal concentration, such as 0.5–1.5% NaCl, which ensures that the ionic strength is in the range in which one or more enzymes in the composition are most active.

[0056] In one embodiment, the composition further comprises a thickening agent.

[0057] In one embodiment, to avoid microbial growth that may impair the composition, the composition further contains a preservative such as a benzoate, propionate, or sorbate, preferably a sorbate such as 0.001 to 10% potassium sorbate. In a preferred embodiment, the composition contains a preservative such as 0.01 to 2% potassium sorbate.

[0058] In one embodiment, the composition contains a soluble calcium salt such as CaCl2 in a concentration of 0.1 to 100 mM, because it is an enzyme activation and stabilization cofactor. In a preferred embodiment, the composition contains a soluble calcium salt such as CaCl2 in a concentration of 1 to 10 mM to stabilize the beneficial enzyme.

[0059] In one embodiment, the composition contains a chaotropic agent such as 0.01 to 8 M urea. This is because it can partially solubilize skin proteins or enhance their swelling, thereby increasing their sensitivity to enzyme activity. In a preferred embodiment, the composition contains 0.1 to 4 M urea to enhance skin swelling and keratin dissolution.

[0060] In one embodiment, the composition contains a reducing agent such as 0.1 to 1000 mM dithiothreitol or glycolic acid. This is because it chemically reduces and breaks down cystine crosslinks that crosslink proteins that may be substrates for the enzyme, thereby increasing their sensitivity to enzyme activity. In a preferred embodiment, the composition contains 1 to 20 mM dithiothreitol or glycolic acid to enhance the reduction and cleavage of disulfide crosslinks in keratin, thereby increasing enzyme substrate reachability.

[0061] In one embodiment, the composition contains an alkali salt such as 1 to 1000 mM NaOH, which allows for pH adjustment to enhance enzyme activity, stability, and substrate solubility. In a preferred embodiment, the composition contains NaOH to enable an alkaline pH composition that enhances skin swelling and keratin solubility.

[0062] In one embodiment, the composition contains an acidic salt such as HCl in a concentration of 1 to 1000 mM, which allows for pH adjustment that enhances enzyme activity, stability, and substrate solubility. In a preferred embodiment, the composition contains HCl to enable an acidic composition that enhances enzyme activity, stability, and a physiologically acceptable pH.

[0063] In one embodiment, the composition contains, preferably in pharmaceutically or cosmetically acceptable concentrations, any chemical and biological agents currently in commercial use, such as benzoyl peroxide, 1,3-benzenediol, azelaic acid, salicylic acid, retinol hormone, and its derivatives, which are used to topically treat follicle-related conditions such as hidradenitis suppurativa and acne forms.

[0064] In one embodiment, the composition contains hyaluronic acid in sufficient quantities to enhance the delivery of enzymes to the skin and its hair follicles.

[0065] In one embodiment, the composition comprises a peptide that enhances the delivery of enzymes to the skin and its hair follicles.

[0066] beauty composition In another embodiment, the composition is a cosmetic composition whose components are of a standard suitable for use in cosmetic compositions, and which, for example, are present in the European Commission's cosmetic ingredient database or can be approved by a regulatory body by obtaining approval from there.

[0067] In this specification and in the claims, a cosmetically acceptable ingredient or an ingredient of suitable specifications for a cosmetic composition means an ingredient having the necessary uniformity and purity for use as a component in the manufacture of a cosmetic composition. Those skilled in the art know how to select such suitable ingredients acceptable for use, for example, by using recognized standards for the cosmetic industry and their legislative measures, such as those of the European Commission or equivalent bodies in the area of ​​the legislative measures. In one embodiment, the cosmetic composition contains the same ingredients and concentrations as those of a pharmaceutical composition, or related but cosmetically acceptable ingredients and concentrations.

[0068] In a particularly suitable embodiment, the present invention is A composition comprising one or more glutamyl endopeptidases capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as keratin, proteins that assist in the function and integrity of their appendages, such as hair follicles, which are assisted by keratinocytes to maintain human skin and its biological barriers, functions, and structures, a. A pharmaceutical composition comprising one or more glutamyl endopeptidases capable of catalyzing the hydrolysis of keratinocyte effector molecules such as keratin, a protein that assists the integrity of human skin and its appendages such as hair follicles, in addition to at least one pharmaceutically acceptable component selected from surfactants that open protein substrates to enzyme activity, agents that promote and stabilize enzyme activity, and agents that enable the enzyme to penetrate the outer layer of the skin, as well as hair follicles and sites of action, thereby enhancing the use and purpose of the composition, or b. A cosmetic composition comprising one or more glutamyl endopeptidases capable of catalyzing the hydrolysis of keratinocyte effector molecules such as keratin, a protein that supports the integrity of human skin and its appendages such as hair follicles, in addition to at least one cosmetically acceptable component selected from surfactants that open protein substrates to enzyme activity, agents that promote and stabilize enzyme activity, and agents that enable the enzyme to penetrate the outer layer of the skin, as well as hair follicles and sites of action, thereby enhancing the use and purpose of the composition. This relates to a composition that is...

[0069] Preferred glutamyl endopeptidases in the above embodiments are glutamyl endopeptidase from Bacillus, glutamyl endopeptidase serine protease bppB from Bacillus pumilus JA16, and glutamyl endopeptidase blaSE from Bacillus licheniformis.

[0070] In general for all embodiments, glutamyl endopeptidase is an endopeptidase having a glutamyl endopeptidase ratio (GR) > 5, where GR is calculated as GR = activity against Suc-AAPE-pNA / activity against the most active Suc-AAP(non)E-pNA, where Suc-AAP(non)E-pNA is one of Suc-AAPA-pNA, Suc-AAPD-pNA, Suc-AAPF-pNA, Suc-AAPI-pNA, Suc-AAPK-pNA, Suc-AAPL-pNA, Suc-AAPM-pNA, Suc-AAPR-pNA, or Suc-AAPV-pNA.

[0071] The aqueous or emulsion composition, which is applied topically or injected into the lesioned skin, preferably further contains at least one surfactant selected from anionic, nonionic, and mixtures thereof, which has compatibility with enzyme stability and activity and enhances enzyme penetration into the outer layers of the skin and delivery to hair follicles and sites of action, thereby enhancing the use and purpose of the composition.

[0072] The suspension composition to be applied topically to the lesioned skin or injected preferably contains a liquid or semi-solid composition in which solid or semi-solid particles, filaments, or mixtures thereof are dispersed. Preferably, the particles have an average size of 200 to 800 nm for topical hair follicle delivery and an average size of 80 to 10000 nm for the injectable composition.

[0073] Preferably, the surfactant that opens the protein substrate to the enzyme activity is selected from Span 20, Span 40, Span 60, Span 80, and Brij 72.

[0074] Preferably, the agent that promotes and stabilizes enzyme activity is selected from glycerol, sugars and propylene glycol, calcium salts, and enzyme inhibitors.

[0075] Preferably, the agent that allows the enzyme to penetrate the outer layer of the skin, as well as the hair follicles and the site of action, thereby enhancing the use and purpose of the composition, is selected from reducing agents such as glycolic acid, chaotropic agents such as urea, and wetting agents such as sorbitan laurate.

[0076] Preferably, the composition according to the present invention is - It is believed that topical application can provide both safe preventive and therapeutic effects, production is commercially efficient, and it is well known to regulatory bodies, therefore it is applied topically, - Unlike topical creams and pressure on irritated skin, microneedle patches are patient / customer friendly / convenient because they limit the enzymes to the target skin area only, so they can be applied by microneedle patches or - Applied by subcutaneous needle injection to treat isolated, injectable lesions / cysts before they can grow further or spread.

[0077] Use of composition for treatment The compositions of the present invention are used to treat skin diseases and hair follicle-related conditions in humans who require such treatment.

[0078] In this application, the term "follicle-related state" means any state relating to the hair follicle, its tissue, cells, intracellular and extracellular proteins and their variants, and the hair shaft and its components, including proteins, carbohydrates, and lipids.

[0079] In one preferred embodiment, uses of the present invention include the medicinal treatment and / or prevention of conditions such as male pattern hirsutism, pseudofolliculitis, acne vulgaris, or acne clusters, pilonidal sinus disease, dissociative cellulitis of the scalp, and tetralogy of follicular obstruction, which includes hidradenitis suppurativa.

[0080] Excessive or pathological hair growth including male pattern hirsutism, hirsutism, or pseudofolliculitis of the pubescent hairs, or skin diseases induced by follicular hyperkeratosis and follicular obstruction, or Tetralogy of follicular obstruction syndrome including acne vulgaris, keratosis pilaris, Dowling-Degos disease, Hailey-Hailey disease, development of mammary duct fistula, Fox-Fordyce disease, trichospinous bunions, atrophic keratosis pilaris, mitotic atrophy, and acne clusters, pilonidal sinus disease, dissociative cellulitis of the scalp, and hidradenitis suppurativa, as well as pustular psoriasis, keratosis pilaris Multiple hair follicle conditions and diseases or hair follicle-related skin diseases, including inflammatory hair follicle-related conditions such as pityriasis, lupus discoidis, lichen planus pilaris, hypertrophic lichen planus pilaris, neoplastic lichen planus, lichen sclerosing, lichen acanthoidis, and Wong-type dermatomyositis; infectious hair follicle-related conditions such as post-kala-azar skin lesions, type I reactions in borderline tuberculous leprosy, and pityriasis pilaris; and diseases of the adnexa resulting in other hair follicle-related diseases such as nevus follicularis, follicular mycosis fungoides, and ranula, are suitable for treatment with the present invention.

[0081] In another preferred embodiment, the use of the present invention includes cosmetic treatment of unwanted hair growth on the human body.

[0082] The compositions of the present invention are used to treat skin diseases and follicle-related conditions by applying the compositions to the skin or lesion area to be treated and allowing the compositions to be present on or in the skin for a retention period that allows the compositions of the present invention to function.

[0083] During treatment, one or more enzymes in the composition of the present invention exert hydrolytic activity on proteins that can be classified as keratin, produced at some point in proliferating or overgrowthed keratinocytes, resulting in the loosening of hair, allowing it to be removed with significantly less force than required to remove untreated hair from the same individual, or even causing it to fall out. Furthermore, the degradation of the hair follicle tissue and its root sheath-specific keratin produced by the proliferating or overgrowthed keratinocytes surrounding the hair shaft results in a reduction in the frequency and / or degree of follicular occlusion / keratin plugging, which limits the progression of follicular occlusion to infected or affected hair follicles. Treatment may also reduce occlusion, resulting in complete keratin removal and rescue of hair follicles in the early stages of diseases such as hidradenitis suppurativa or related syndromes, thereby potentially controlling and preventing early-stage disease progression, recurrence, and onset, as well as the hallmark lesions that follow more severe stages of the disease, which place an even greater burden on the patient.

[0084] The compositions of the present invention can be injected or even applied topically to more severely diseased areas of the body, including obstructed and / or infected hair follicles or skin cysts, and in this embodiment, can be used to reduce or intervene in disease progression to prevent further progression of the disease in one or more areas.

[0085] The retention time must be long enough to allow one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules, such as the protein keratin, which aids in the integrity, function, and structure of the biological barrier of human skin and its appendages, such as hair follicles, to have a sufficient effect. Typically, the retention time for a topically applied composition is selected within a range of 2 minutes to 24 hours, e.g., 3 to 60 minutes, e.g., 5 to 30 minutes, e.g., overnight. In some embodiments, for example, if the composition is a gel, the retention time will actually last until the enzymes become inactive. Enzyme inactivation will occur, for example, in a gel composition, after several hours to several days to several weeks, when the skin, mucous membrane, or hair follicle tissue naturally desquams and the enzymes are pushed out, and / or when the outside of the skin is cleaned. In some embodiments, for example, if the composition is a medical patch or other non-liquid composition that can be completely removed by physical peeling or washing, the retention time will actually last until the enzymes become inactive. Enzyme inactivation may occur after several hours to several days, for example, in a medical patch composition. For example, in some embodiments where the composition is an intra-injectable agent, the retention time is not important.

[0086] Those skilled in the art can assess the integrity of the ORS of hair follicles and lesional tissue with hair shafts still present using the simple technical concepts described in Figure 2 and Example 3. The force required to remove a hair is a convenient functional and quantitative measure that can be used by those skilled in the art using the well-known technique described in Figure 2. When the force required to remove a treated hair is compared to the force required to remove an untreated hair, it is preferable to measure several individual hairs and calculate the average force to compensate for variations in hair that will occur due to various skin biologies and skin types, their current state, and the natural hair follicle cycle in which hair grows, falls out, and regrows over time. Accordingly, according to the present invention, the force required to remove a treated or untreated hair should preferably be calculated as the average of at least three or more hairs in a skin biopsy. If the number of hairs available in a skin biopsy is less than that, it is preferable to calculate the average based on all hairs available in the biopsy. Preferably, the force required to remove the treated hair is reduced by at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, or at least 70%, or at least 80%, compared to untreated hair, or to a degree that corresponds to pain reduction to at least painless hair removal or enhanced prevention or control of hair follicle-related diseases.

[0087] Example 3 shows an example of a method for measuring the force required to remove hair. The integrity of the orthostatic stenosis (ORS) in human skin can be further evaluated by tissue imaging after incubating plucked human hairs with ORS with enzymes. The histological integrity of the ORS and surrounding tissues is visually inspected by those skilled in the art using microscopy and staining specific to the tissue and cellular components. Examples 1-10 and Figures 3-7 show methods for evaluating differences that those skilled in the art may consider useful for the purposes of the present invention, which include treating follicular dermatological diseases and follicular-related conditions.

[0088] In one embodiment, the composition of the present invention is used to treat male pattern hirsutism and hirsutism. In this embodiment, the present invention provides an efficient treatment that enables the removal of excess hair shafts, including their roots, from the affected skin area, and provides a painless or pain-reduced treatment with a longer-lasting effect.

[0089] In another embodiment, the composition of the present invention is used to treat pseudofolliculitis. In this embodiment, the present invention provides an efficient treatment that allows for the removal of excess hair from affected skin areas, particularly of the face and neck, or to alleviate skin irritation caused by morphological abnormalities in the hair that are likely to be caused by dysfunction of the hair follicle anchoring tissue, cells, and their proteins, and damaging the skin.

[0090] In another embodiment, the composition of the present invention is used to treat acne vulgaris. In this embodiment, the present invention provides an efficient treatment with a low risk of skin irritation, enabling the reduction of acne, papules, nodules, cysts, pustules, lesions, and the removal of excessive hair from the affected skin area.

[0091] In another embodiment, the composition of the present invention is used to treat keratosis pilaris, in which blocked hair follicles have resulted in widespread, itchy nodules.

[0092] In another embodiment, the compositions of the present invention are used to treat follicular hyperkeratosis and obstruction-related diseases such as Dowling-Degos disease, Hailey-Hailey disease, and mammary duct fistula.

[0093] In another embodiment, the compositions of the present invention are used to treat hidradenitis suppurativa and other tetralogy of hair follicles, or follicular occlusion disorders in which the condition develops depending on the same follicular keratin-related development. In this embodiment, the present invention is particularly suited to treating early lesion development in which the compositions of the present invention are used to prevent or reduce follicular occlusion before the presentation of later, more severe symptoms. Since all patients with the described hidradenitis suppurativa and related disorders have recurrent lesions that are the result of the hair follicle cycle throughout the growth phase, the present invention is effective for all stages of the condition or disease severity.

[0094] In another embodiment, the compositions of the present invention are used to treat inflammatory follicle-related conditions including pustular psoriasis, pityriasis rubra pilaris, lupus discoid, lichen planus pilaris, hypertrophic lichen planus pilaris, neoplastic lichen planus, lichen sclerosing, lichen acanthoid, and Wong-type dermatomyositis; infectious follicle-related conditions including post-kala-azar skin lesions, type I reactions in borderline tuberculous leprosy, and pityriasis pilaris; and other follicle-related diseases including comedone nevi, follicular mycosis fungoides, and ranula.

[0095] The present invention is further described and supported by the following embodiments provided solely for the purpose of illustrating the invention, and should not be considered limiting in any sense. [Examples]

[0096] [Example 1] Screening of endopeptidases for ORS tissue activity by hair release This example describes an assay to screen a panel of different endopeptidases for the ability of endopeptidases to release hair from the skin, providing a measure of catalytic activity in the hydrolysis of proteins such as keratin that anchor hair, which aids in the function and integrity of hair follicles.

[0097] The experimental design is shown in Figure 1. Pig ears are a good physiological model of human hairy skin, but it is necessary to control for innate biological variability. Ears were obtained postmortem as samples from one source of landrace pigs bred for animal experiments, rinsed thoroughly with water, placed in pairs in airtight plastic bags, and immediately frozen at -20°C. Ears were used in experiments less than one month after freezing. For each enzyme experiment, a pair of frozen ears were thawed for approximately one hour in 250 mL of 150 mM physiological NaCl (20°C) in a plastic bag, ensuring that the skin was completely covered during thawing. The salt solution was discarded, and the ears were thoroughly washed for two minutes with 100 mL of shampoo solution (4 mL of MacUrth Mild Organic Shampoo, containing 96 mL of water, with disodium lauryl sulfosuccinate (anionic) surfactant and coconut oil alkyl glucoside (nonionic) surfactant). The ear was rinsed in tap water, dried with a paper towel, and then the skin was dissected.

[0098] To best isolate biological variability from the effects of the enzyme, mirror images of the ear pair used in each experiment were used for the reference feed and enzyme samples (1). To eliminate differences in absolute effects between pairs, negative and positive controls were included in all ears. The positive control was glutamyl endopeptidase blaSE from Bacillus licheniformis, which showed the greatest effect during the assay design phase, and this data point allowed for verification that the biological samples were reliable and consistent between ear pairs. In each experiment, four 20 × 20 mm skin pieces per ear, i.e., eight skin pieces per pair, were carefully cut from the dorsal side of the ear, and the skin was carefully removed from the cartilage by cutting alongside the cartilage surface with a sharp scalpel.

[0099] The assay was performed at a skin surface temperature of 30°C using 25 mM Tris / HCl, 150 mM NaCl, 5 mM CaCl2, 2 mM potassium sorbate, and 1% Span 20 (HZ buffer and negative control) buffered to a neutral pH of 7.4. These conditions were selected to stabilize the enzyme with calcium ions, reduce microbial growth during the experiment, promote wetting, and limit irritation, respectively, to make them suitable for pharmaceutical and cosmetic compositions.

[0100] The 11 endopeptidases screened are listed in Table 1 and represent a broad diversity of endopeptidase classes with different proteolytic specificities, publicly annotated in the UniProt or MEROPS databases. These were individually purified by column chromatography to a purity in which a single band could be observed after SDS-PAGE separation and Coomassie blue staining.

[0101] [Table 1]

[0102] Two skin samples were incubated as negative controls (2400 μl HZ buffer per skin sample), two skin samples were incubated as positive controls (2400 μl HZ buffer and 100 ppm HZ-2 per skin sample), and four skin samples were incubated as endopeptidase (HZ of (1)) incubation (2400 μl HZ buffer and 100 ppm endopeptidase per skin sample). The skin samples were incubated at 30°C for 3 hours. The solution was discarded, the skin samples were thoroughly rinsed with tap water and dried using paper towels, and then left under a fume hood for 1 hour.

[0103] 50 x 25 mm pieces of sports tape (Sportsdoc Medical Pro Deluxe, 25 mm, Svea medical sports AB, obtained from Sweden) were gently placed on each skin piece in the same direction as the hair growth, and pressed firmly with sufficient force to ensure contact between the hair and the adhesive (2). The tape was peeled off in a single motion in the opposite direction to the natural hair growth (3). Because the adhesive of the sports tape is very strong, the hair will either break and the hair root will remain in the skin, or the tape will pull the hair out while leaving the hair root sheath intact. After peeling off the tape, the tape was stained with 1% paradimethylaminocinnamaldehyde (DMACA) in 0.5 M HCl, which stains the IRS bright red (4). The number and length of the red IRS are a convenient means of identifying the capacity of endopeptidases that catalyze the hydrolysis of proteins such as keratin that fix the hair, which aids in the function and integrity of the hair follicle. After taking photographs, the IRS was evaluated by counting the number of red IRS and by measuring the total length of IRS present on the tape using drawing software (OpenOrienteering Mapper) with the tape width to normalize the data. The results are listed in Table 2 below. To account for variability between ear pairs, the relative number of IRS and the relative total length of IRS were calculated as follows: [(enzyme-negative control) / (positive control-negative control)] × 100%.

[0104] [Table 2]

[0105] We identified glutamyl endopeptidase blaSE, derived from Bacillus licheniformis, as the best-performing endopeptidase among 11 tested endopeptidases, and it most effectively catalyzes the hydrolysis of anchor proteins such as keratin, which support hair follicle function and integrity.

[0106] To determine whether glutamyl endopeptidase is particularly well-suited for catalyzing keratin, which supports hair follicle function and integrity, three glutamyl endopeptidases from other organisms (see Table 3) were studied as described above.

[0107] [Table 3]

[0108] The results for the three additional glutamyl endopeptidases tested in the assay described above are listed in Table 3 and are shown in Table 4 below.

[0109] [Table 4]

[0110] From the table above, it can be seen that glutamyl endopeptidase is the most suitable endopeptidase for catalyzing the hydrolysis of proteins such as keratin that fix hair, thereby supporting the function and integrity of hair follicles.

[0111] [Example 2] Definition of glutamyl endopeptidase activity. This example describes an assay for evaluating whether an endopeptidase is a glutamyl endopeptidase in the context of the present invention. A glutamyl endopeptidase is an endopeptidase that cleaves the carboxyl terminus of a glutamic acid residue (or an aspartic acid residue in a phosphate buffer), i.e., glutamyl endopeptidases have preference for negatively charged amino acid residues at the P1 position of the substrate. The following assay was used to test whether the endopeptidases that showed a significant response in Example 1, which are included in SEQ ID NOs: 1-4, 7-8, 10, and 12-14 listed in Tables 1 and 3 and used in the present invention, are glutamyl endopeptidases.

[0112] 20 μl of each endopeptidase was diluted with 0.01% Triton X-100 and placed in wells in a microtiter plate at 25°C, and assays were performed using the following commercially available substrates (Bachem AG, Bubendorf, Switzerland): Suc-AAPA-pNA (Bachem 4015680), Suc-AAPD-pNA (Bachem 4018122), Suc-AAPE-pNA (Bachem 4017343), Suc-AAPF-pNA (Bachem 4002299), Suc-AAPI-pNA (Bachem 4017698), Suc-AAPK-pNA (Bachem 4017329), Suc-AAPL-pNA (Bachem 4003646), Suc-AAPM-pNA (Bachem 4006760), Suc-AAPR-pNA (Bachem 4017320), Suc-AAPV-pNA (Bachem 401767).

[0113] The reaction was initiated by adding 200 μl of pNA substrate (50 mg, dissolved in 1.0 ml of DMSO and further diluted 75-fold in 50 mM Tris / HCl, 0.01% Triton X-100, pH 8.0). The microtiter plate was read with a Molecular Devices VERSAmax microtiter reader, and the first increase in OD405 was a measure of endopeptidase activity. If a linear plot was not obtained after a 4-minute measurement time, the endopeptidase was diluted and the assay was repeated.

[0114] The results for the 10 endopeptidases tested in the above assay are shown in Table 5 below. The data correspond to the relative activity of each endopeptidase for 10 different Suc-AAPX-pNA substrates (X = different amino acids), i.e., the activity of a specific Suc-AAPX-pNA substrate divided by the activity of the most active Suc-AAPX-pNA substrate among the 10. Endopeptidase dilutions were eliminated in the calculations.

[0115] [Table 5]

[0116] Table 5 shows that glutamyl endopeptidase blaSE (HZ-2) from Bacillus licheniformis, glutamyl endopeptidase II sprE (HZ-13) from Streptomyces glyceus, serine protease bppB (HZ-15) from Bacillus pumilus JA16, and V8 protease (HZ-35) from Staphylococcus aureus exhibit the highest activity against the Suc-AAPE-pNA substrate, while showing extremely low relative activity against other substrates. In conclusion, these endopeptidases are considered to be glutamyl endopeptidases. To evaluate whether an endopeptidase is a glutamyl endopeptidase, the inventors defined a glutamyl endopeptidase ratio (GR) calculated as follows: GR = activity against Suc-AAPE-pNA / activity against the most active Suc-AAP(non)E-pNA.

[0117] The glutamyl endopeptidase according to the present invention is defined as an endopeptidase with a glutamyl endopeptidase ratio (GR) > 5.

[0118] [Example 3] Quantitative determination of the hair release effect by local treatment with endopeptidase This example describes an assay for quantifying the hair-releasing effect of a selected endopeptidase by measuring the force required to pull out hairs after topical application of the enzyme composition to the hairy skin of a pig.

[0119] To separate biological variability between skin samples from the effects of enzymes, the same considerations as those described in Example 1 were taken in this example, with the following additional considerations: perforations were made in the dermal layer (5), different ear positions were used (6), and the molds were glued to the skin to ensure precise local interaction.

[0120] Figure 2 shows a schematic diagram of the experimental design. To eliminate variations in the thickness of the superficial layer of the pig ear skin and to increase the delivery of endopeptidase to the hair follicles in the deeper layers of the skin, the dorsal side of the ear was treated with a derma roller (RoHS-compliant Argador derma roller system with 540 × 1.5 mm microneedles, approved for cosmetic use in the European Union) by rolling the derma roller over the skin four times in vertical, horizontal, and two diagonal directions under strong pressure to ensure even perforation of the skin. Three square molds made of polymethyl methacrylate (PMMA) were attached to the dorsal side of each ear in a mirror image position using ethyl cyanoacrylate adhesive to define small 18 × 18 mm skin areas for enzyme treatment.

[0121] The physiological temperature, pH, and salt concentration of the composition were the same as in Example 1, and 10 mM DTT (HZ buffer DTT) was added. Six PMMA squares in a pair of ears were used for three different types of treatment: negative control, positive control, and endopeptidase samples (HZ of (6)). Nine endopeptidases, including SEQ ID NOs: 1, 3, 6-8, 10, and SEQ ID NOs: 12-14, which showed a positive response in Example 1 and are listed in Tables 1 and 3, were included in this example.

[0122] Two squares were negative controls (600 μl HZ buffer DTT per square), two squares were positive controls (600 μl HZ buffer DTT and 100 ppm HZ-2 per square), and two squares were endopeptidase-treated samples (600 μl HZ buffer DTT and 30 ppm–100 ppm endopeptidase per square). Skin samples with squares and enzymes were stored horizontally at 4°C for approximately 20 hours, leaving the ear intact. The skin samples were then incubated at 30°C, equal to the skin surface temperature, for 4 hours.

[0123] The square solution was discarded, and the square was removed from the skin by cutting along the inside of the square with a scalpel, releasing the hairs from the adhesive. The treated skin area was carefully washed in deionized water, and the skin pieces were carefully dried with paper towels. Finally, the force required to pull individual hairs from each skin piece after enzyme incubation was measured by fixing the hairs to a Sauter FK 10 digital force gauge connected to a spindle motor with a vertically moving string and clamp (7).

[0124] result: A summary of the results is shown in Table 6 below. The decrease in force compared to the negative control was calculated as follows: ((Negative control - enzyme) / (Negative control)) × 100%. The decrease in force compared to the positive control was calculated as follows: ((Negative control - enzyme) / (Negative control - Positive control)) × 100%. For each skin sample, the force of at least 7 and up to 10 individual hairs per sample was measured, and the mean was calculated for two sets of samples for each treatment on a pair of ears. Due to variability between pairs of ears, data on the decrease in force compared to the positive control were only accepted if the positive control (HZ-2 at 100 ppm) was considered significantly lower than the negative control with a p-value of 0.05 or less using Student's t-test, and are shown in the table below.

[0125] [Table 6]

[0126] Table 6 shows that at 100 ppm, HZ-2 (positive control), a glutamyl endopeptidase blaSE from Bacillus licheniformis, showed the greatest reduction in potency compared to the negative control, while HZ-10, HZ-11, HZ-15, and HZ-35 at 100 ppm were only able to reduce potency to 79-86% of HZ-2 at 100 ppm, which is equivalent to HZ-2 at only 30 ppm (85%). HZ-13 showed lower performance than expected from its performance in Example 1, but in Example 4, it became clear that its performance was unstable under the specific conditions tested in this example. [Example 4] Stability of endopeptidase activity.

[0127] This example describes an assay for evaluating whether endopeptidase is stable under the skin application conditions tested in the present invention. One relevant skin application is the screening assay described in Example 1. Another relevant skin application is the skin assay described as Example 3.

[0128] Two incubation buffers were used in this stability assay. Incubation buffer A: 25 mM Tris / HCl, 150 mM NaCl, 5 mM CaCl2, 2 mM potassium sorbate, 1% Span 20, pH 7.4 (used in Example 1), and Incubation buffer B: 25 mM Tris / HCl, 150 mM NaCl, 5 mM CaCl2, 2 mM potassium sorbate, 10 mM DTT, 1% Span 20, pH 7.4 (used in Example 3).

[0129] Each endopeptidase was transferred to two incubation buffers A and B by diluting to 0.10 mg / mL (at least 20-fold dilution), and small aliquots were stored on ice until activity analysis. The majority of the dilution in incubation buffer A was incubated at 30°C for 3 hours (similar to the incubation of skin samples in Example 1). After incubation, the residual activity of the endopeptidase was determined for dilution A incubated at 30°C and for the corresponding dilution maintained on ice (0°C). The stability of Example 1 was determined by dividing the activity of dilution A incubated at 30°C by the activity of dilution A incubated at 0°C.

[0130] The majority of the dilution of incubation buffer B was incubated at 30°C for 4 hours. Since enzyme inactivation is minimal at low temperatures such as 4°C, the additional overnight storage at 4°C in Example 3 had little effect on the residual activity. After incubation, the residual activity of the endopeptidase was determined. The stability of Example 3 was determined by dividing the activity of dilution B incubated at 30°C by the activity of dilution A incubated at 0°C. In this way, the inventors eliminate both the decrease in stability due to DTT in the buffer and the decrease in stability due to longer incubation times.

[0131] [Table 7]

[0132] Appropriate substrates for each endopeptidase are listed in Table 7, using either Suc-AAPX-pNA (where X represents a different amino acid in different substrates) or Protazyme AK. For the Suc-AAPX-pNA substrate, residual activity was determined by the same assay as described in Example 2 of the present invention. For Protazyme AK, residual activity was determined by adding 20 μl of endopeptidase solution (diluted in 0.01% Triton X-100) to a 1.5 mL Eppendorf tube containing 500 μl of Protazyme AK suspension (one Protazyme AK tablet from Megazyme Ltd., Wicklow, Ireland, suspended in 2.0 ml of 0.01% Triton X-100) and 500 μl of ice-cold mixture of 50 mM MOPS / NaOH, 0.01% Triton X-100, pH 7.0. The Protazyme AK assay was initiated by transferring the tubing to a pre-warmed Eppendorf thermomixer set to 37°C. The tubing was then incubated in the thermomixer at 1100 rpm for 15 minutes. The tubing was returned to the ice bath. 200 μl of the cooled supernatant was transferred to a microtiter plate, and the OD405 was read using a Molecular Devices VERSAmax microtiter reader. The difference between the OD405 of the endopeptidase sample and the OD405 of the buffer blank was used as a measure of the residual activity of the endopeptidase sample. If the measured OD405 value exceeded 1.5 OD405 units, the endopeptidase was further diluted and the assay was repeated.

[0133] The data corresponding to the relative activity of each endopeptidase was calculated as described above and listed in Table 8, taking dilution factors into consideration.

[0134] [Table 8]

[0135] It can be seen that all enzymes were stable under the same conditions as in Example 1. Therefore, the enzymes remained active throughout the entire incubation period.

[0136] It can be seen that not all enzymes were stable under the same conditions as in Example 3. This is because HZ-8, HZ-11, HZ-13, and HZ-18 showed decreased activity after being placed in incubation buffer B at 30°C for 4 hours, suggesting that these endopeptidases were not 100% active throughout the entire incubation period. These data explain why HZ-13 did not function well as a glutamyl endopeptidase in Example 3.

[0137] [Example 4] Stability of endopeptidase activity. This example describes an assay for evaluating whether the endopeptidase is stable under the skin application conditions tested in the present invention. One relevant skin application is the screening assay described in Example 1. Another relevant skin application is the skin assay described in Example 3.

[0138] [Table 9]

[0139] Two incubation buffers were used in this stability assay. Incubation buffer A: 25 mM Tris / HCl, 150 mM NaCl, 5 mM CaCl2, 2 mM potassium sorbate, 1% Span 20, pH 7.4 (used in Example 1), and Incubation buffer B: 25 mM Tris / HCl, 150 mM NaCl, 5 mM CaCl2, 2 mM potassium sorbate, 10 mM DTT, 1% Span 20, pH 7.4 (used in Example 3).

[0140] Each endopeptidase was transferred to two incubation buffers A and B by diluting to 0.10 mg / mL (at least 20-fold dilution), and small aliquots were stored on ice until activity analysis. The majority of the dilution in incubation buffer A was incubated at 30°C for 3 hours (similar to the incubation of skin samples in Example 1). After incubation, the residual activity of the endopeptidase was determined for dilution A incubated at 30°C and for the corresponding dilution maintained on ice (0°C). The stability of Example 1 was determined by dividing the activity of dilution A incubated at 30°C by the activity of dilution A incubated at 0°C.

[0141] The majority of the dilution of incubation buffer B was incubated at 30°C for 4 hours. Since enzyme inactivation is minimal at low temperatures such as 4°C, the additional overnight storage at 4°C in Example 3 had little effect on the residual activity. After incubation, the residual activity of the endopeptidase was determined. The stability of Example 3 was determined by dividing the activity of dilution B incubated at 30°C by the activity of dilution A incubated at 0°C. In this way, the inventors eliminate both the decrease in stability due to DTT in the buffer and the decrease in stability due to longer incubation times.

[0142] Appropriate substrates for each endopeptidase are listed in Table 9, using either Suc-AAPX-pNA (where X represents a different amino acid in different substrates) or Protazyme AK. For the Suc-AAPX-pNA substrate, residual activity was determined by the same assay as described in Example 2 of the present invention. For Protazyme AK, residual activity was determined by adding 20 μl of endopeptidase solution (diluted in 0.01% Triton X-100) to a 1.5 mL Eppendorf tube containing 500 μl of Protazyme AK suspension (one Protazyme AK tablet from Megazyme Ltd., Wicklow, Ireland, suspended in 2.0 ml of 0.01% Triton X-100) and 500 μl of an ice-cold mixture of 50 mM MOPS / NaOH, 0.01% Triton X-100, pH 7.0. The Protazyme AK assay was initiated by transferring the tubing to a pre-warmed Eppendorf thermomixer set to 37°C. The tubing was then incubated in the thermomixer at 1100 rpm for 15 minutes. The tubing was returned to the ice bath. 200 μl of the cooled supernatant was transferred to a microtiter plate, and the OD405 was read using a Molecular Devices VERSAmax microtiter reader. The difference between the OD405 of the endopeptidase sample and the OD405 of the buffer blank was used as a measure of the residual activity of the endopeptidase sample. If the measured OD405 value exceeded 1.5 OD405 units, the endopeptidase was further diluted and the assay was repeated.

[0143] result: The data corresponding to the relative activity of each endopeptidase was calculated as described above and listed in Table 10, taking dilution factors into consideration.

[0144] [Table 10]

[0145] It can be seen that all enzymes were stable under the same conditions as in Example 1. Therefore, the enzymes remained active throughout the entire incubation period.

[0146] It can be seen that not all enzymes were stable under the same conditions as in Example 3. This is because HZ-8, HZ-11, HZ-13, and HZ-18 showed decreased activity after being placed in incubation buffer B at 30°C for 4 hours, suggesting that these endopeptidases were not 100% active throughout the entire incubation period. These data explain why HZ-13 did not function well as a glutamyl endopeptidase in Example 3.

[0147] [Example 5] Glutamyl endopeptidase has only slight activity against human skin outer layer proteins. This example describes an assay for evaluating how aggressive endopeptidases are against the superficial layers of the body's skin, including the major biological barriers to the delivery of enzymes or other pharmaceutical compounds to the skin and hair follicles.

[0148] In Example 1, nine endopeptidases, numbered SEQ ID NOs: 1, 3, 6-8, 10, and 12-14, listed in Tables 1 and 3, showed target activity against ORS tissue. In this example, the degree of hydrolysis of stratum corneum proteins was analyzed for each enzyme.

[0149] Hardened, thickened skin consists of an enlarged layer of dead keratinized cells on the surface of the skin, i.e., the stratum corneum. From a health perspective, this is either a defense response typically seen on the palms and soles of the feet, or it may be the cause of pathology in skin conditions. Hardened substrates in granular or powder form were prepared using a metal scraper on the dry soles of healthy human volunteers' feet. The substrates were prepared by removing soluble proteins using two washes with 0.01% Triton X-100. A 25 mg / mL suspension in 0.01% Triton X-100 was prepared, and 200 μL of the suspension was pipetted into a 1.5 mL Eppendorf tube. The substrate was precipitated by short-time centrifugation, and the supernatant was discarded. The pellet was resuspended in 200 μL of 0.01% Triton X-100 by stirring with an Eppendorf thermomixer (2000 rpm, 2 minutes), the suspension was centrifuged again, and the supernatant was discarded. The second precipitate was resuspended in 180 μL of HZ buffer by Eppendorf mixing to prepare a 190 μL suspension, which was placed on ice. The endopeptidase was diluted in 0.01% Triton X-100 to 500 ppm, and 10 μL of each dilution was added to Eppendorf tubes containing the suspended skin matrix to obtain two sets of endopeptidase with a final concentration of 25 ppm for the assay. The Eppendorf tubes were incubated in a preheated Eppendorf thermomixer (30 minutes, 30°C, 2000 rpm), and after incubation, the tubes were returned to ice and cooled for several minutes. The tubes were centrifuged, and the supernatant was diluted 10-fold in 0.01% Triton X-100.

[0150] The degree of hydrolysis in the diluted supernatant for each endopeptidase was expressed as the concentration of solubilized protein by measuring the absorbance at 562 nm using the PIERCE BCA protein assay kit (Thermo Scientific 23227). The results are shown in Table 11 below.

[0151] [Table 11]

[0152] Table 11 shows that HZ-10, HZ-11, and HZ-19 hydrolyzed most of the dermal layer. Excessive nonspecific hydrolysis is undesirable because it can also break down biological barriers to non-therapeutic enzymes. Under these conditions, glutamyl endopeptidases exhibited hydrolytic activity levels of 1 / 4 to 1 / 5 of that of skin proteins (HZ-2, HZ-15, HZ-13) and nearly zero (HZ-35). The limited dermal layer activity of the enzymes included in this invention may be an advantage because these enzymes are not only desired to disrupt ORS to promote hair release, but also to have some selective activity to promote enzyme delivery to hair follicles, hair follicles occluded with stratum corneum cells, and keratin debris, or to delivery to hair follicle-related lesions.

[0153] [Example 6] Glutamyl endopeptidase strongly destroys human ORS tissue in human beard hair. This example describes how the intrinsic selectivity of glutamyl endopeptidase leads to the degradation of keratin components in human ORS tissue and hair follicle-related diseases. Two endopeptidases, SEQ ID NO: 1 (HZ-2) and SEQ ID NO: 7 (HZ-10), listed in Table 1, which showed activity in Examples 1 and 3 and were stable in Example 4, were selected for microscopic analysis in this example, and their histological activity after enzyme incubation was evaluated.

[0154] Freshly plucked beard hairs were collected to form a sample of 10 hairs, which were fixed on a glass slide (Figure 3). A total of six samples were prepared. One sample was left untreated, directly stained, and imaged (8). The other hair samples were incubated in the following solutions: One sample was a negative control (200 μl of 50 mM Tris, pH 8, 2 mM CaCl2 buffer (9)), two samples were incubated with HZ-2 (1 ppm HZ-2 in 200 μl of the same buffer (10), or 30 ppm HZ-2 in 200 μl of the same buffer (11)), and two samples were treated with HZ-10 (1 ppm HZ-10 in 200 μl of the same buffer (12), or 30 ppm HZ-10 in 200 μl of the same buffer (13)). The glass slides were covered to prevent evaporation and incubated at 30°C for 3 hours. Hairs were stained with red 1% DMACA in 0.5 M HCl IRS stain, as well as purple / blue ORS stain containing a 1:1 mixture of McIlvane buffer, 2.5 mg / ml toluidine blue in pH=3.6, and Walpoles buffer, and 0.1% rhodamine B in pH=4.4, and immediately imaged at 40x magnification using a light microscope. From 2-3 representative hairs in each sample in Figure 3, it can be seen that the ORS of the negative control (9) was no different from that of the untreated control (8). In incubation with 30 ppm HZ-10 (13), only completely keratinized hair shafts remained after incubation, while incubation with 1 ppm HZ-10 (12) showed IRS that was still keratinized but not very resistant. In incubation with 30 ppm HZ-2 (11), the interior of the ORS was visible after incubation, while in incubation with 1 ppm HZ-2 (10), the ORS tissue was only sufficiently hydrolyzed to swell to twice the size of the negative control. The data suggest that, compared to HZ-10, HZ-2, despite its narrower specificity, potently destroys ORS in a less aggressive manner in terms of cell and tissue destruction. Selective tissue destruction is preferred in this invention because it limits undesirable side effects and ensures a safer procedure. [Example 7] Glutamyl endopeptidase strongly hydrolyzes type I and type II keratin in human ORS tissue.

[0155] This example describes glutamyl endopeptidase protein substrates in human ORS tissue. The endopeptidase (HZ-2) of Sequence ID No. 1, listed in Table 1, which showed a positive response in Examples 1 and 3, was stable in Example 4, and exhibited significant activity in Examples 5 and 6, was analyzed using SDS-PAGE to investigate its ability to specifically hydrolyze proteins in ORS tissue.

[0156] Freshly plucked beard hairs were collected and prepared as a sample consisting of five strands in an Eppendorf tube. A total of three samples were prepared (Figure 4). The samples were incubated in buffer at 30°C for 30 minutes. One sample was used as a negative control (50 mM Tris / HCl, pH 8, 0.1% Triton X-100, and 2 mM CaCl2 buffer), one sample was incubated with HZ-2 (1 ppm in the same buffer, 200 μL), and one sample was incubated with HZ-2 (5 ppm in the same buffer, 200 μL). After incubation, the supernatant was carefully removed, and the remaining tissue was dissolved in 8 M urea, 2% SDS, 100 mM DTT, and 100 mM Tris / HCl, pH 8 at 30°C for 30 minutes. Due to the poor solubility of IRS and hair shafts, the majority of the extracted proteins originated from ORS, which was confirmed by observing the absence of ORS under a microscope before and after lysis (data not shown). Proteins were concentrated using a 10 kDa molecular cutoff spin filter, separated using SDS-PAGE, and stained with Coomassie blue to visualize the proteome. MES buffer and a 10% Bolt Bis-Tris Plus gel were used. Results are shown in Figure 4. The approximate actual size corresponding to the bands on the gel was identified using the PageRuler Plus Prestained Protein ladder (14) (ThermoFischer Scientific). Negative control (15), 1 ppm HZ-2 (16), and 5 ppm HZ-2 (17) were equally loaded. Two strong band staining groups were found, which those skilled in the art would conclude correspond mainly to type I (50-60 kDa) and type II (60-70 kDa) keratin in ORS. Under these conditions, only 1 ppm of HZ-2 degraded type I keratin, while 5 ppm of HZ-2 completely differentiated type I and type II during the experiment, leaving multiple bands intact in both cases. The results indicate that HZ-2 is a potent and selective ORS keratinase.Type I and Type II keratins of ORS are necessary for hair fixation, are effector molecules for hyperkeratosis and occlusion of hair follicles, and support hair follicle function and integrity. The selective hydrolysis of these by HZ-2 explains the hair release observed in Examples 1 and 3, as well as how compositions of glutamyl endopeptidases such as HZ-2 can be used to treat hair follicle-related conditions and hair follicle system skin diseases.

[0157] [Example 8] Glutamyl endopeptidase selectively destroys ORS tissue in healthy human skin. This example illustrates the selective destruction of ORS tissue by glutamyl endopeptidase in human skin. Two endopeptidases, SEQ ID NO: 1 (HZ-2) and SEQ ID NO: 7 (HZ-10), listed in Table 1, which were able to release hair in Examples 1 and 3, were stable in Example 4, and showed significant activity in Examples 5 and 6, were analyzed by histological evaluation after enzyme incubation.

[0158] Six mm punch biopsies of axillary skin from healthy female volunteers were frozen-sectioned into 10 μm slices using a microtome. Skin slices were placed on glass slides for microscopy. Three representative, adjacent skin slices cut transversely through the hair follicles were prepared and their overview is shown in Figure 5 at 40x magnification (18). The tissue slices were incubated at 30°C for 30 minutes in a buffer droplet on the slide and covered to prevent evaporation, and visualized at 400x magnification using a light microscope from the circled area in (18) around the hair shaft and ORS. One slice served as a negative control (50 mM Tris / HCl, pH 8, and 2 mM CaCl2 buffer (19)), the next slice was incubated with HZ-2 (100 ppm HZ-2 in the same buffer (20)), and the following slice was incubated with HZ-10 (100 ppm HZ-10 in the same buffer (21)). After incubation, the slices were stained with hematoxylin and eosin (18-21), or with 2.5 mg / ml toluidine blue in McIlvane buffer, pH=3.6, and 0.1% rhodamine B in Walpoles buffer, pH=4.4 for 10 minutes, followed by staining with 1% rhodamine B in Walpoles buffer, pH=4.4 for 1 minute (22-24). The data shown in Figure 5 reveals that HZ-2(20) selectively destroys ORS tissue and forms cell aggregates (black arrows), suggesting primarily intercellular ORS effects compared to the negative control (gray arrows), while leaving the dermis intact. However, HZ-10(21) destroys the contents of ORS cells while clearly maintaining the ORS tissue intact, suggesting primarily intracellular ORS effects (black arrows), but clearly eliminating nuclei present in the dermal collagen (gray arrows).Using a 200x fluorescence microscope, the contrast was enhanced when the rhodamine B stain was excited with green light. Under these conditions, HZ-2 (23, white arrow in ORS) also did not significantly destroy keratinized non-ORS tissues of the epidermis ((22, 23) whiter than (24)), such as IRS ((23, 24) white arrow) and epidermis (not shown), compared to the negative control (22, black arrow in ORS) and HZ-10 (24, white arrow in ORS). This suggests that HZ-2 had significantly lower off-target activity than HZ-10 while effectively destroying ORS tissue. The effects observed with HZ-2 are most desirable for the effective and safe treatment of follicle-related conditions and follicular cutaneous diseases, as described in the compositions and uses of the present invention.

[0159] [Example 9] Glutamyl endopeptidase selectively destroys ORS tissue in pathological hidradenitis suppurativa skin. This example describes the selective destruction of ORS tissue by glutamyl endopeptidase in the skin of patients with hidradenitis suppurativa. Two endopeptidases, SEQ ID NO: 1 (HZ-2) and SEQ ID NO: 7 (HZ-10), listed in Table 1, which showed a positive response in Examples 1 and 3, were stable in Example 4, and exhibited significant activity in Examples 5-6 and 8, were used to treat patient tissue, which was then analyzed by histological evaluation after enzyme incubation. 10 × 10 × 8 mm axillary lesion skin excision biopsies (Figure 6, (25)) obtained from female volunteers with moderate hidradenitis suppurativa exhibiting clear inflammatory features were completely suspended in either a negative control (125 mM Tris / HCl pH 8, 0.25% Triton X-100, and 5 mM CaCl2 buffer) or HZ-2 (500 ppm HZ-2 in the same buffer) and incubated at 30°C for 3 hours. The specimens were fixed in 10% formalin for one week, then dried using vacuum and embedded in paraffin. After placing the skin appendages on a glass slide using a microtome, the specimens were sectioned into 4 μm slices, deparaffinized by thawing at 60°C for one hour, and washed with Tissue Clear, 99% ethanol, 70% ethanol, and deionized water. After re-fixing the tissues overnight with Bouin's fixative, the specimens were stained, thoroughly washed with deionized water, stained with fresh Weigert's iron hematoxylin for 10 minutes, washed with deionized water, counterstained with 10% Sirius Red picric acid for 15 minutes, and dehydrated twice in 99% ethanol.

[0160] Figure 6 shows 200x zoom fluorescence microscope images of two representative slices per treatment condition, after excitation with Sirius Red. These images reveal that a negative control pre-lesional follicle with traces of keratinization (26) occluded the follicular cyst (27), while HZ-2 selectively destroyed the ORS tissue in two pre-lesional follicles (28), leaving the ORS cells isolated around the hair shaft and the surrounding dermis intact. HZ-2 also demonstrated the degradation of early-stage follicular cysts (29), leaving them empty. In conclusion, similar to the ORS in healthy patients in Example 8, HZ-2 is active in the lesional ORS tissue of patients with hidradenitis suppurativa, and possibly in pre-cystic ORS tissue, and therefore is likely to have the same effect on the tissues of patients suffering from multiple follicle-related conditions and follicular system skin diseases, which are the targets of treatment by the compositions and use of the present invention. [Example 10] Glutamyl endopeptidase promotes local delivery of protein to hair follicles.

[0161] This example demonstrates that compositions containing HZ-2 promote the delivery of high molecular weight compounds, such as proteins (in this case, the enzyme itself), to hair follicles, as indicated by the increased penetration of reflective nanoparticles into the hair follicles of porcine hairy skin. Two endopeptidases, SEQ ID NO: 1 (HZ-2) and SEQ ID NO: 7 (HZ-10), listed in Table 1, which showed a positive response in Examples 1 and 3, were stable in Example 4, and exhibited significant activity in Examples 5-6 and 8-9, as well as tissues, were analyzed by reflective confocal laser microscopy.

[0162] In Figure 7, a buffer containing 25 mM Tris / HCl, pH 8, 150 mM NaCl, 1% Span 20 surfactant, 2 mM potassium sorbate, and 5 mM CaCl2 was used as a negative control. The buffer was very similar to the composition used in the localized experiment in Example 3, except that it lacked DTT. The negative control, HZ-2 (500 ppm HZ-2 in the same buffer), and HZ-10 (500 ppm HZ-10 in the same buffer) were added strictly topically to the dorsal side of pig ears without prior microneedle puncture, as shown in Figure 2(6) and Example 3. Skin sections were incubated at 4°C for 20 hours, and then incubated at 30°C for 5 hours inside a PMMA rectangle attached with adhesive. After discarding the buffer and removing the PMMA rectangle, the resulting enzyme-treated pig skin samples were kept on ice for up to two hours until analysis began.

[0163] Hair was trimmed to a length of 3 mm, and 120 nm silica-cored gold nanoparticles (GNP) (CE612960, SEB-250, Sebacia Inc.), with peak absorbance at 800 nm, were rubbed into the skin for 2 minutes using gloved fingers. GNP penetration was used as a reporter for the activity of ORS enzymes in the skin and hair follicles by analysis using an 830 nm reflection-type confocal laser microscope en face (Figure 7). As GNP penetrates the hair follicles, GNP enhanced the contrast of the microscopic image by increasing the reflectance around the hair in the image. Scans were performed at 0 μm without GNP addition (30) and with GNP addition (31). During the experiment, the depth of GNP penetration for each sample was evaluated by measuring the reflectance at 188 μm (32) and 300 μm (33) below the skin surface.

[0164] In the negative control (37), GNP was found to penetrate only to a depth of 188 μm (34) and not to 300 μm. Notably, HZ-2 enabled GNP to penetrate to a depth of 300 μm, which corresponds to the majority of the hair follicle infundibulum and hair follicle-related sites of action to which the enzyme needs to be delivered to treat hair follicle-related skin diseases such as hidradenitis suppurativa (35, 38). However, the more nonspecific HZ-10 could not penetrate beyond 188 μm (36) and left no trace of GNP at a depth of 300 μm (39). 300 μm is the technical limit of confocal laser microscopy, which suggests that HZ-2 penetrates even deeper. This example further illustrates HZ-2's ability to increase the delivery of nanoparticles to hair follicles, or in some cases transfollicular delivery, which is a desirable means for formulating enzyme-based and other agents for treating hair follicle-related diseases. This example further illustrates the potential use of HZ-2 to enhance the permeability of other low and high molecular weight compounds into hair follicles, including proteins such as the HZ-2 enzyme itself, or glutamate-specific HZ-2 or any other protein that is stable in the presence of any pharmaceutical product for which locally delivered products have an industrial benefit.

Claims

1. A composition comprising one or more enzymes capable of catalyzing the hydrolysis of keratinocyte effector molecules such as the protein keratin, which assists the function and integrity of human skin and its appendages, such as hair follicles, which are assisted by keratinocytes to maintain biological barriers, their function and structure, a. A pharmaceutical composition comprising one or more enzymes in addition to at least one pharmaceutically acceptable component. or b. A cosmetic composition comprising one or more enzymes in addition to at least one cosmetically acceptable ingredient. A composition that is

2. The composition according to claim 1, wherein one or more enzymes, selected from glutamyl endopeptidases, are capable of catalyzing the hydrolysis of keratinocyte effector molecules such as the protein keratin, which are assisted by keratinocytes to maintain the integrity of human skin and its appendages, such as hair follicles, in order to maintain biological barriers, functions, and structures.

3. The composition according to claim 2, wherein the glutamyl endopeptidase is selected from glutamyl endopeptidases having at least 60% sequence identity to the polypeptide having the sequence of Sequence ID No. 1, for example, at least 70% sequence identity, for example, at least 80% sequence identity, for example, at least 90% sequence identity, for example, at least 95% sequence identity, for example, at least 96% sequence identity, for example, at least 97% sequence identity, for example, at least 98% sequence identity, for example, at least 99% sequence identity.

4. The composition according to claim 2, wherein the glutamyl endopeptidase is selected from glutamyl endopeptidases having at least 60% sequence identity to the polypeptide having the sequence of Sequence ID No. 13, for example, at least 70% sequence identity, for example, at least 80% sequence identity, for example, at least 90% sequence identity, for example, at least 95% sequence identity, for example, at least 96% sequence identity, for example, at least 97% sequence identity, for example, at least 98% sequence identity, for example, at least 99% sequence identity.

5. The composition according to claim 3, wherein the glutamyl endopeptidase is selected from glutamyl endopeptidase having an amino acid sequence containing or consisting of SEQ ID NO: 1, or a variant thereof having one or more substitutions compared to SEQ ID NO: 1, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

6. The composition according to claim 4, wherein the glutamyl endopeptidase is selected from glutamyl endopeptidase having an amino acid sequence comprising or consisting of SEQ ID NO: 13, or a variant thereof comprising one or more substitutions compared to SEQ ID NO: 13, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

7. The composition according to any one of claims 2 to 6, wherein glutamyl endopeptidase is present in an amount ranging from 1 μg to 10 mg of enzyme protein per gram of the composition.

8. The composition according to any one of the claims, which is formulated as a gel, nanoemulsion and microemulsion, and semi-solid emulsions of hydrophilic and hydrophobic phases of semi-solid and / or liquids containing two or more phases, such as creams, cloths, sponges, pastes, pills, shampoos, soaps, jellies, lotions, foams, ointments, film formulations, sprays, liquids, liquid formulations, oils, ointments, suspensions of nanometer- to micrometer-sized particles in liquid or solid suspensions, medical patches, medical patches with microneedles, medical patches with soluble microneedles, derma rollers with microneedles, iontophoresis, compressed air, solid skin implants, semi-solid skin implants, liquid skin implants, skin tattoos, roll-ons, swabs, syrups, tapes, wafers, or as an injectable solution, injectable solution, emulsified injectable, or suspension injectable, which promotes the delivery of enzymes to the site of action and enhances their function and purpose.

9. A medical device comprising the pharmaceutical composition according to any one of claims 1 to 8.

10. The medical composition according to claim 9, wherein the medical device is a patch or injection solution containing the composition.

11. Use of the composition according to any one of claims 1 to 8 or the medical device according to claim 9 or 10 for treating hair follicle-related skin diseases and hair follicle-associated conditions.

12. The composition is a pharmaceutical composition, and the hair follicle-related conditions and hair follicle-related skin diseases include follicular occlusion, keratosis pilaris, Dowling-Degos disease, Hailey-Hailey disease, mammary duct fistula, or acne clusters, pilonidal sinus disease, dissociative cellulitis of the scalp, and hidradenitis suppurativa, which result in the development of hair follicle-related skin diseases or acne vulgaris; or pustular psoriasis. The use according to claim 11, selected from pityriasis rubra pilaris, lupus discoidis, lichen planus pilaris, hypertrophic lichen planus pilaris, neoplastic lichen planus pilaris, lichen sclerosing, lichen acanthoidis, inflammatory follicle-related conditions including Wong-type dermatomyositis, post-kala-azar skin lesions, type I reaction in borderline tuberculous leprosy, infectious follicle-related conditions including pityriasis pilaris; and other follicle-related diseases including comedone nevi, follicular mycosis fungoides, and ranula.

13. Use of the composition according to any one of claims 1 to 8 or the medical device according to claim 9 or 10 for treating hair follicle-related skin diseases and hair follicle-associated conditions.

14. The use according to claim 11 or 12, wherein the composition is used to prevent the development of hair follicle-related skin diseases and hair follicle-associated conditions in susceptible individuals.

15. The use according to claim 11, wherein the composition is a cosmetic composition and is used for the removal of unwanted hair.

16. The use according to claim 11, wherein the composition is a cosmetic composition and is used for skin peeling.

17. The use according to claim 11 or 12, wherein the composition is used to facilitate the skin and hair follicle delivery of one or more proteins, such as enzymes, which themselves are contained therein.

18. The use according to claim 11 or 12, wherein the composition is used to enhance the delivery of other pharmaceutical or cosmetic ingredients to hair follicles and skin.

19. A composition according to any one of claims 1 to 8, for use as a pharmaceutical or drug for treating human hair follicle-related conditions and hair follicle-related skin diseases.

20. Male-pattern hirsutism, hirsutism, or pseudofolliculitis of the pubescent region; or follicular keratosis induced by follicular hyperkeratosis, or development of follicular skin diseases or acne vulgaris, including follicular occlusion, keratosis pilaris, Dowling-Degos disease, Hailey-Hailey disease, mammary duct fistula, or acne clusters, pilonidal sinus disease, dissociative cellulitis of the scalp, and hidradenitis suppurativa; or pustular psoriasis, pityriasis rubra pilaris, lupus discoides, hair The compositions according to claims 1 to 8 for use in the treatment of inflammatory follicle-related conditions, including lichen planus porosus, hypertrophic lichen planus pilaris, neoplastic lichen planus, lichen sclerosis, lichen acanthoides, Wong-type dermatomyositis, post-kala-azar skin lesions, type I reaction in borderline tuberculous leprosy, and infectious follicle-related conditions, including pityriasis follicularis; and other follicle-related diseases, including nevus of the comedone, follicular mycosis fungoides, and ranula.

21. A composition according to any one of claims 1 to 6 for cosmetic use relating to human hair follicle-related conditions and hair follicle-related skin diseases.

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